Nucleotide sequences for detection of Bacillus anthracis

ABSTRACT

The invention provides purified and isolated DNA fragments from  Bacillus anthracis  chromosomal DNA, primer sets and probes derived therefrom, as well as kits and detection methods for  B. anthracis . The methods of the invention provide for specific detection of anthrax over closely related strains of  Bacillus , as well as accurate detection of low numbers of  B. anthracis  in an environmental sample containing large amounts of non-specific DNA. The invention is applicable to food, health care, and military applications.

This application is a division of application Ser. No. 09/879,027, filedJun. 12, 2001, now U.S. Pat. No. 6,448,016.

GOVERNMENTAL INTEREST

The invention described herein may be manufactured, used, and licensedby or for the U.S. Government.

FIELD OF THE INVENTION

The present invention relates generally to the detection of Bacillusanthracis. More specifically, the invention relates to anthrax-specificpolymorphic signature sequences identified on, and isolated from theanthrax chromosome DNA for use as B. anthracis-specific markers. Primersets and hybridization probes designed from these DNA fragmentsequences, as well as amplification of the fragments, can be used in avariety of platforms for anthrax detection.

BACKGROUND OF THE INVENTION

Anthrax—primarily a disease of herbivorous animals but of rareoccurrence in humans—is caused by Bacillus anthracis. Cutaneous anthraxis acquired via injured skin or membranes, entry sites where the sporegerminate into vegetative cells. Proliferation of vegetative cellsresults in gelatinous edema. Alternatively, inhalation of the sporesresults in high fever and chest pain. Both types can be fatal unless theinvasive aspect of the infection can be intercepted. Bacillus anthracisis a biological warfare (BW) agent. Ten grams of anthrax spore can killas many people as a ton of the chemical warfare agent, sarin. Due to thehighly lethal nature of anthrax and BW agents in general, there is greatneed for the development of sensitive and rapid BW agent detection.Current detection technology for biological warfare agents havetraditionally relied on time-consuming laboratory analysis or onset ofillness among people exposed to the BW agent.

In theory, the use of specific antibodies or distinguishing DNA probesare the two approaches to modernizing detection technology in thisfield. However, antibody-based detection of threat agents suffers fromdrawbacks. For example, interference from other environmentalcontaminants precludes detection, or detection limits of current levelsfail to meet the detection thresholds set by governmental testingprotocols. Alternatively, the threat agent, such as with anthrax spore,may be poorly immunogenic.

Since a sample suspected of containing a BW agent like B. anthraciscould contain such a small yet lethal amount of spores, and anoverwhelming amount of other interfering materials, the ability toamplify the agent's genomic material affords a choice of target sitesfor developing signature probes for specific detection of that agent.Development of highly discriminating techniques are crucial to achievingthe stated goals of rapid and sensitive BW detection.

Current PCR-based detection methods of B. anthracis rely on the use ofprimers amplifying tripartite exotoxin genes and/or the polyglutamiccapsule genes (Jackson et al, Proc. Natl. Acad. Sci, 95: 1224-9 (1998)).Both sets of genes comprise virulence factors and are located on the twoindigenous plasmids of anthrax bacteria, pXO1 (174 kbp; toxin) and pXO2(95 kbp; capsule). Under normal conditions, the two plasmids in B.anthracis do not move across the related bacilli of the “B. cereusgroup” (which is comprised of B. anthracis, B. cereus, B. thuringiensisand B. mycoides (although B. mycoides does not produce toxin andtherefore may be grouped differently from the other three members)).However, under certain conditions, these plasmids are known to betransferred from B. anthracis to B. cereus and B. thuringiensis (Ruhfelet al, J. Bact., 157: 708-11 (1984)). Yet B. cereus and B. thuringiensiscontaining one or both of these plasmids do not cause anthrax.Therefore, detection of anthrax based solely on virulence factors cangive rise to a false-positive determination.

Two chromosomal DNA fragment sequences from B. anthracis have beenpreviously identified and used in identifying the presence of B.anthracis bacteria. One, designated Ba813, is a 277 bp long DNA fragment(Patra et al., FEMS Microbiol. 15: 223-231 (1996)) and the other, vrrA,is a region of sequence variability containing variable repeats (caa tatcaa caa) (Anderson et al., J. Bacteriol. 178: 377-384 (1996)).

Additionally, Yamada et al (U.S. Pat. No. 6,087,104) identified uniqueregions of the DNA gyrase sub-unit B (gyrB) gene for each of the closelyrelated bacteria of the B. cereus group, and designed oligonucleotideprimers corresponding to those unique regions for amplification-baseddetection methods. However, amplification of DNA segments unique to eachof the B. cereus group members occurred only when the correct targetstrain DNA by itself was present in the PCR protocol.

However, since the development of more rapid and more sensitive BWdetection methodologies is of such importance to the military as well aspublic health sectors of the U.S. government, there is great need tocontinue the process of identifying, cloning, and sequencing ofpolymorphic DNA markers from chromosomal DNA of threat agents. With thispurpose in mind, comprehensive libraries of BW agent-specific signaturesequences can be built, and from there useful diagnostic primers andprobes can be designed for highly discriminating detection methods. Thepresent invention as herein described fulfills these objectives.

SUMMARY OF THE INVENTION

Accordingly, it is an embodiment of the invention to provide purifiedand isolated DNA sequences from the chromosomal DNA of B. anthracis, asshown in FIGS. 1a-1 f and corresponding to SEQ ID NOS: 1-7.

Another embodiment of the invention is the use of these sequences indiagnostic assays to accurately analyze samples for environmentalcontamination by B. anthracis spores and for the early diagnosis ofanthrax in human and non-human animals. In this embodiment resides thecapability not only to distinguish over closely related Bacillusspecies, thereby affording more sensitive and reliable detection foranthrax, but also to detect a very minuscule concentration of anthraxspores in the presence of an overwhelming amount of unrelatednon-specific environmental DNA.

In yet another embodiment, the invention provides for primer pairs (SEQID NOS: 8-15) designed from the aforementioned purified and isolated DNAsequences of the first embodiment. The primer pairs are useful incarrying out PCR amplification-based detection of B. anthracis.Alternatively, the invention further provides hybridization probesdesigned from the novel isolated DNA sequences of the first embodiment,such probes being useful in a number of assay platforms for B.anthracis.

Kits useful in the practice of the invention are also provided,containing at least one container comprising at least one pair ofprimers or at least one hybridization probe specifically designed toselectively amplify or bind, respectively, chromosomal DNA of B.anthracis.

These and other embodiments of the invention will be better appreciatedby the following detailed disclosure.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1a depicts one of the subject chromosomal DNA fragments (985 bp)isolated from B. anthracis and referred to as 280FR (SEQ ID NO: 1).

FIG. 1b depicts one of the subject chromosomal DNA fragments (711 bp)isolated from B. anthracis and referred to as 173F (SEQ ID NO: 2).

FIG. 1c depicts one of the subject chromosomal DNA fragments (722 bp)isolated from B. anthracis and referred to as 290BF (SEQ ID NO: 3).

FIG. 1d depicts one of the subject chromosomal DNA fragments (754 bp)isolated from B. anthracis and referred to as 173R (SEQ ID NO: 4).

FIG. 1e depicts one of the subject chromosomal DNA fragments (932 bp)isolated from B. anthracis and referred to as 248FR (SEQ ID NO: 5).

FIG. 1f depicts the left (bp 1-440) (SEQ ID NO: 6) and right (bp718-985) (SEQ ID NO: 7) flanking regions of the chromosomal DNA fragmentreferred to as 280FR (FIG. 1a), which flanking regions are adjacent to apreviously identified B. anthracis fragment Ba813.

FIGS. 2a, 2 b, 3 a, and 3 b collectively depict developedelectrophoretic gels of the RAPD amplification profile using randomprimers and genomic DNA isolated from:

1) Agrobacterium tumifaciens; 2) Bacillus thurigiensis; 3) B. cereus; 4)B. anthracis Δ Sterne (pOX1⁻-pOX2⁻); 5) B. anthracis Δ Ames(pOX1⁻/pOX2⁺); and 6) B. anthracis Ames (wild-type, pOX1⁺/pOX2⁺).Primers are indicated below the gel.

FIG. 4 depicts the developed electrophoretic gel of the RAPDamplification profile using random primers of anthrax-specific DNA inthe presence of different amounts of non-specific environmental sludgeDNA.

FIG. 5 depicts the developed electrophoretic gel showing the detectionlimit of anthrax-specific DNA probes in spiked environmental samplescontaining from 0.001-1.0 ng of anthrax DNA.

FIG. 6 depicts the results of amplification of anthrax-specific DNA fromspiked soil/sludge sample using the high-fidelity primer sets accordingto the invention.

DETAILED DESCRIPTION OF THE INVENTION

The process of identifying genus-specific signature sequences orpolymorphic loci can be approached in a number of ways. One approachentails the cloning of genomic material from, e.g., related bacteria,followed by laboriously eliminating the common shared regions among therelated organisms. Such an approach was utilized by Patra et al inidentifying anthrax chromosome fragment, Ba813.(FEMS Microbiol. 15:223-231 (1996)). Alternatively, the process of identifying polymorphicloci and the development of diagnostic probes or primers can be greatlysimplified by using RAPD (randomly amplified polymorphic DNA), aPCR-based technology (Williams et al, Nucleic Acids Research, 18(22):6531-35 (1994)). In this technique, small amounts of DNA are subjectedto PCR using a single oligonucleotide of random sequence as a primer.The amplification products are resolved on agarose or polyacrylamidegels giving rise to a pattern that is strain specific. Many of theproducts generated by RAPD-PCR are derived from repetitive DNAsequences. As these sequences are frequently species-specific, RAPD-PCRis potentially a quick method for developing species-specific diagnosticPCR primers and probes.

Accordingly, in the present invention, random sequence primers of, e.g.,10 nucleotide bases (ten-mers), are used under low-stringency annealingtemperatures with genomic DNA from a given BW agent such as B. anthracisand its close relatives in PCR protocols. The number and size ofamplified fragments derived from genomic DNA of a desired BW agent underthese conditions depend on the existing number of priming sites and thedistance between the priming sites in opposite direction on the twostrands of DNA. In practice, the number and size of amplified fragmentsdepend on the ability of a single primer to anneal to complementarysites on the two strands in opposite directions (5′→3′ and 3′→5′) within˜2500 bp of each other. Identification, cloning, and obtaining ofsequence information from polymorphic DNA markers located on thechromosomal DNA enables the formation of a library of agent-specificsignature sequences, also referred to as a DAF pattern DNA AmplificationFingerprinting pattern). As between the closely-related members of agroup such as B. cereus RAPD produces a common sub-set of amplified DNAfragments since the genomes are largely similar. However, there will bea further sub-set of amplified fragments unique to each individualmember on account of inherent DNA polymorphisms.

A general outline and brief description of the methods utilized in thepresent invention in order to elucidate polymorphic DNA markers from thechromosomal DNA of B. anthracis follows. A more detailed description isprovided in the “Examples” section.

The Isolation and Screening of DNA from Anthrax and Related BacteriaAgainst Random Primers in PCR Protocols

DNA from wild-type B. anthracis strain Sterne, and DNA from twoplasmid-free strains of B. anthracis, and a strain containing only thepXO2 plasmid were prepared following standard procedures known to one ofordinary skill for isolation of genomic DNA. The DNA's were diluted to aconcentration of 1 ng/ml, and the majority of DNA was in the size rangeof about 30-50 kbp. As a control, DNA from other related bacilli, B.cereus, B. thuringiensis and B. subtilis were also prepared in likemanner and included. For initial screening purposes only, DNA from aunrelated bacterium, Agrobacterium tumifaciens, was also included.

Three hundred (300) random decamer (10-mer) primers were purchased froma commercial source (University of British Columbia, Vancouver, BC,Canada). RAPD-PCR amplification reactions were conducted in routinemanner on the DNA of the several strains B. anthracis and on the DNA ofthe related bacilli.

The Analysis of PCR Amplified DNA Through Agarose Gel Electrophoresis

The PCR amplified DNA was analyzed through agarose gel electrophoresisand the DAF pattern was photographed and documented. Primers thatamplified unique-size DNA fragments from wild-type anthrax strain andplasmid-free strain were identified. After several rounds ofamplification, primers amplifying consistent DNA fragments from anthraxDNA were selected.

Fragment Amplification at the Preparatory Level

The fragments identified in the preceding step were amplified at apreparatory level and purified following gel electrophoresis. Five DNAfragments were then cloned in pTA cloning vector (from Invitrogen Co.,CA) for sequencing.

Nucleotide Sequencing and Computer Analysis

The nucleotide sequence for each of the five cloned DNA fragments wasdetermined following the manufacturer's protocol based on the well-knowndideoxy sequencing method of Sanger et al (Proc. Natl. Acad Sci, 74:5463-67 (1977)) using an automated dideoxy sequencer (Perkin-Elmer,Applied Biosystems Division, Foster City, Calif.). Computer analysis ofeach of the DNA sequences was performed using MacVector, version 6.0(Oxford Molecular Ltd., Oxford).

The five DNA fragment sequences from anthrax chromosome are 280FR (FIG.1A) (SEQ ID NO: 1), 173F (FIG. 1B) (SEQ ID NO: 2), 290BF (FIG. 1C) (SEQID NO: 3), 173R (FIG. 1D) (SEQ ID NO: 4), and 248FR (FIG. 1E) (SEQ IDNO: 5). The sequences represented by SEQ ID NO: 2 through SEQ ID NO: 5did not match any known sequence in the Genebank database. A region of277-bp in sequence 280FR (SEQ ID NO: 1), from bp 441 to bp 717 was foundto be homologous to Ba813, an anthrax-specific chromosomal regionidentified by Patra et al., supra. Accordingly, it is not an embodimentof the present invention to seek protection for the 277-bp region of280FR by itself. Nor is it an embodiment of the invention to seekprotection of any primer or probe derived or designed exclusively fromwithin bp 441 to bp 717, inclusive. However, the sequences of each ofthe two regions ((FIG. 1F) bp 1-bp 440 (SEQ ID NO: 6) and bp 718-bp 985(SEQ ID NO: 7)) that flank the 277-bp region were not found to match anyknown sequence in the Genebank database. Accordingly, it is intendedthat the flanking regions represented by SEQ ID NO: 6-7 are part of theprotection sought herein. Consequently, any primers and probes derivedin whole or in part (i.e., primers and probes derived from the junctureof 5′ or 3′ end of Ba813 with either flanking region) from these regionsare also contemplated.

As will become evident in the examples set forth hereinbelow, the fiveDNA fragment sequences of the invention (SEQ ID NO: 1-5) are unique intheir ability to discern between anthrax DNA and the DNA of theremaining members of the B. cereus family. Hence the five sequencesprovide a novel “blueprint” for design of numerous primer pairs andhybridization probes useful for diagnostic applications.

Primers

A number of primer sequences were designed from the cloned 280FR, 173F,290BF, 173R, and 248FR DNA sequences. These primer sequences aregenerally from 10 to 30 bases in length, more preferably, from 15 to 20bases in length. The primers were designed using MacVector, version 6.0(Oxford Molecular Ltd., Oxford) and primer synthesis was carried out byLife Technologies, Inc. (Gaithersburg, Md.). Primers were designed toamplify fragments of B. anthracis chromomsomal DNA in the size range offrom about 130 to 550 base pairs, preferably from 300 to 500. Arepresentative number of primers according to the invention are asfollows:

Primers Ref. Name 5′-CAT TCG GTG TTT TTT GAC GAG C-3′ R173F1 (SEQ ID NO:8) 5′-CTT TGC AGA AGC ATT AGC AGA AGG-3′ R173B22 (SEQ ID NO: 9) 5′-TGTTCC AAG AAT GAA GCG TAC TCC-3′ R290F1 (SEQ ID NO: 10) 5′-TGA AGC CTA CTCCCG TTT CAA G-3′ R290F4 (SEQ ID NO: 11) 5′-TCA CCG TTA GAA TCA CGC CACC-3′ R290B11 (SEQ ID NO: 12) 5′-GCC AAA ACA TTT ATC GTC CCA G-3′ R290B17(SEQ ID NO: 13) 5′-CAA TGG GTT GAT ACT CAC AGT CCA G-3′ F290F1 (SEQ IDNO: 14) 5′-CCT TGC TGC AAC ATA TAC CCC ATA G-3′ F290B20 (SEQ ID NO: 15)

The above primers were paired as a forward and reverse primer set togive the following expected fragment size during amplification:

Expected Primer Combination Fragment Size (base-pair) R173F1 & R173B22(SEQ ID NO: 8 & 9) 390 R290F1 & R290B11 (SEQ ID NO: 10 & 12) 330 R290F4& R290B17 (SEQ ID NO: 11 & 13) 360 F290F1 & F290B20 (SEQ ID NO: 14 & 15)520

Primers according to the invention may optionally have a detectablelabel or tag conjugated thereto. Suitable labels or tags are well-knownto those working in the field, and, for example, may be chosen toprovide a radioactive, calorimetric, fluorometric or luminescent signaldepending on the particular application. Incorporation of an appropriatevisualization label into custom-synthesized primers and probes followsroutine protocol of the DNA synthesizer employed. It is within thepreferred scope of the invention, for example, that the primers hereindescribed be synthesized to incorporate a fluorescent tag so thatdetection of anthrax organism can be carried out on a Taq-man® platformor other suitable diagnostic medium.

Gene Probe Approach

Unique fragments selected from the five B. anthracis chromosomal DNAfragments shown in SEQ ID NO: 1-5 can be synthesized in large quantitiesthrough polymerase chain reaction, and conjugated to any solid support,e.g., glass or silica beads, multiwell plate, dipstick, or the like. Theconjugated fragments are rendered single-stranded according towell-known chemical treatment and are used as hybridization robes fordetecting anthrax in an environmental sample suspected of contamination.

Alternatively, probes consisting of much smaller DNA fragments can beprepared from amplification products obtained using the primer sets orpairs described above. The fragments of this embodiment are fromapproximately 100 to 500 bp in length, and more preferably from 170 to350 bp. A probe designed to detect amplified products of anthraxchromosomal DNA sequence is preferably designed not to hybridize to thesequence of the primers used for amplification. In such an application,it is preferable that the probe be designed to detect amplificationproducts by hybridizing to sequences between the primer sequences.

In the practice of anthrax detection methods using the probes of theinvention, at least one signature probe hybridizing with specificity toamplified anthrax chromosomal DNA is detectably labeled. For example, atleast one probe is labeled with a biotin moiety and/or at least oneprobe labeled with a fluorescently labeled probe. The amplified DNAfragments are then bound to a solid support such as a bead, multiwellplate, dipstick, or the like that is coated with streptavidin. Thepresence of bound amplified DNA fragments can be detected using anantibody with fluorescent tag conjugated to alkaline phosphatase orhorseradish peroxidase. The enzymatic activity of luminescent orfluorimetric substrate, or conversion of the substrate (such as pNPP foralkaline phosphatase) to product can also be used to detect and/ormeasure the presence of B. anthracis PCR products.

The amplification-based method for detection of B. anthracis in a samplecomprises selecting at least one pair of primers derived from thenucleotide sequences represented by SEQ ID NO: 1-7, which primer pair(s)is/are specific for B. anthracis DNA but not which does not recognize,or anneal to DNA from the related strains B. cereus, B. thuringiensis,and B. subtilis. The primers are then mixed with a sample containingnon-specific DNA and suspected of containing DNA from B. anthracis.Using standard PCR, amplification is carried out on any DNA to which theprimers in the previous step have annealed. If any amplification productis formed it is subjected to analysis by separation and detected usingsuitable detection means. As mentioned supra, the primers used in thepresent amplification-based methods are not derived exclusively fromwithin the region of bases-pairs spanning 441 to 717, inclusive, of SEQID NO: 1. Moreover, the same proviso applies to any probe derived fromSEQ ID NO: 1.

A further embodiment of the present anthrax detection methods is aimedat increasing the specificity for recognition of B. anthracis to theexclusion of other related strains of the B. cereus family, therebyreducing the likelihood of false positive indications. As discussedsupra, the plasmids of B. anthracis, which carry the virulence factorsTOX gene and CAP gene, can transfer under certain conditions to theother related members of the family. Although present in these relatedstrains, the virulence factors do not cause anthrax infection.Accordingly detection methods for anthrax based on virulence factorsalone are not adequate. Hence, by employing one or more primer pairs orone or more hybridization probes of the invention in conjunction withprimers specifically amplifying TOX gene and CAP gene of B. anthracis indetection protocols, the likelihood of false positive indications isreduced. Thus, a true positive indication for anthrax would require notonly detectable amplification products of TOX and CAP genes, but also,and more dispositively, detectable amplification product from B.anthracis chromosomal DNA.

The present invention lends itself readily to the preparation of “kits”containing the elements necessary to carry out detection methods for B.anthracis. Such a kit may comprise a carrier being compartmentalized toreceive in close confinement therein one or more container, such astubes or vials. One of the containers may contain unlabeled ordetectably labeled DNA primer pairs or one or more hybridizationprobe(s). The labeled DNA primers may be present in lyophilized form orin an appropriate buffer as necessary. One or more containers maycontain one or more enzymes or reagents to be utilized in PCR reactions.These enzymes may be present by themselves or in mixtures, inlyophilized form in appropriate buffers.

In an alternate embodiment, the kit would contain at least one containerof optionally detectably labeled primers or probes according to theinvention, and at least one container with e.g., a primer pairspecifically amplifying or a hybridization probe for TOX gene and CAPgene of B. anthracis.

Finally, the kit may contain all of the additional elements necessary tocarry out the technique of the invention, such as buffers, extractionreagents, enzymes, pipettes, plates, nucleic acids, nucleosidetriphosphates, filter paper, gel materials, transfer materials,autoradiography supplies and the like.

EXAMPLES

The following examples are intended solely to illustrate one or morepreferred aspects of the invention and are not to be construed aslimiting the scope of the invention.

Materials and Methods

Soil and Sludge Source:

Activated sludge was obtained from Back River water treatment plant inDundalk, Md., and used for isolation of genetic material. The soilsamples were collected from farm area around Harford county, MD, and USArmy APG, Edgewood Arsenal, Edgewood, Md.

Anthrax DNA and Random Primer Source:

DNA from wild-type B. anthracis strain Sterne, was kindly provided byDr. Tim Hoover, USAMRAID, Fort Dietrich, Md. DNA from other B. anthracisstrains, such as VNR1-Δ1 and ASterne, both plasmid-free; and ΔAmes,pXO2+were prepared in our laboratory following standard procedures forisolation of genomic DNA. The DNA was diluted to a concentration of 1ng/ml, and the majority of the DNA was in the size range of about 30-50kbp. DNA from other related bacilli, B. cereus, B. thuringensis, B.subtilis, and Agrobacterium tumifaciens were prepared following similarprocedures. The random primers (10-mer) were purchased from commercialsources (University of British Columbia, Vancouver, BC, Canada).

Isolation and Purification of DNA:

DNA from the environmental samples was worked up using a ‘Soil DNAIsolation Kit’ from MoBio Laboratory, Inc. (CA, USA). Two grams ofsoil/sludge was processed in three replicates according to themanufacturer's protocol. Approximately 500-1000 ng total DNA wasrecovered, and much of DNA was ≧30-40 kbp in size.

RAPD-PCR Amplification:

Routine PCR reactions were carried out in a final volume of 10 or 20 μlusing 96-well tray in GeneAmp PCR System (Perkin Elmer-Cetus). Thereaction mix contained I-30 ng of DNA, 200 μM dNTPs, 2 mM MgCl₂, and 0.5unit of Taq DNA polymerase, and 0.2 μM of selected random decamer (IO-mer) primers (Rastogi and Cheng, 1997).

DNA Analysis:

The amplificates following each PCR run were mixed with 6× loading dyein a ratio of 5:1 (DNA:dye). The samples were electrophoresced through1.4% agarose gel submerged in 1×TAE buffer (Sambrook et al. In:Molecular cloning—A laboratory manual, 2^(nd) ed., CSH Lab Press, CSH,NY (1980)) using constant voltage of 100 volts. The DNA in gels wasstained with ethidium bromide (0.5 μg/ml) and destained beforephotographing using Polaroid film 667.

DNA Seguencing:

Automated DNA sequencer model 373 (Perkin Elmer, Applied BiosystemsDiv., Foster City, Calif.) was used for DNA sequencing. After subcloningof the amplified DNA fragment into pCR-blunt vector (Invitrogen,Carlsbad, Calif.), both strands of cloned DNA fragments were sequencedfollowing manufacturer's protocol. Computer analysis of the DNA wasperformed with MacVector program (Oxford Molecular Ltd., Oxford, UK).

Quality of DNA from Soil/Sludge:

Soil and sludge contain large number of diverse microorganisms, as wellas genetic material released from decaying organisms. Presence of thehumus material (humic acids, humates or salts of humic acid, fulvicacids and fulvates, lignite and humin) interferes with detection,measurement, and routine molecular analysis of the DNA (Thurman et al.“Isolation of soil and aquatic humic substances”, In: Eds. F H Frimmeland R F Christman, Humic substances and their role in the environment, JWiley & Sons, Ltd., NY, 1988, 31; Tsai et al, Detection of low numbersof bacterial cells in soils and sediments by polymerase chain reaction,AEM, 58: 754 (1992). DNA isolated using ‘MoBio Soil DNA Kit’ wasamenable to restriction digestion and ligation (results not shown).These results establish that the commercially available kit is aneffective procedure for removal of humus material from environmentalDNA.

Example 1 Amplification of Anthrax-Specific DNA in Spiked Samples

The inventors herein have identified five random 10-mer primers,designated as 173, 248, 280, 290, and 361, result in amplification ofDNA fragments from the chromosomal DNA of wild-type anthrax. Theseprimer did not amplify the similar-sized DNA fragments from the genomicDNA from strains related to anthrax, i.e., B. cereus, B. thuringiensis,and an unrelated strain Agrobacterium tumifaciens (see FIGS. 2a through3 b). In addition, genomic DNA from wild-type anthrax and two derivativestrains lacking one or both plasmids were used as target DNA. The factthat similar-sized DNA fragments are observed in the three anthraxstrains, indicates that the priming sites must be located on thechromosome. The amplification pattern derived from A. tumifaciens DNAwas very different from Bacillus DNA, and therefore, this DNA was notincluded in subsequent experiments.

As shown in FIG. 4, anthrax-specific fragments were amplified fromspiked environmental sludge DNA samples, when primer nos.173, 248, 280,and 290 were used. The spiked samples included anthrax DNA at aconcentration of 0.01 and 0.1 ng and sludge DNA mixed at 10, 50, 100, or200 ng concentration. In general, presence of 200 ng of sludge DNAresulted in failure of the primers to amplify anthrax-specific DNAfragments. Further, primer no. 280 failed to amplify anthrax-specificDNA fragments even at 50 or less ng of sludge DNA. However, primer nos.173, 248, and 290 clearly demonstrate the specific ability of theserandom primers to amplify the anthrax-specific DNA fragments in sludgesamples spiked with anthrax DNA.

Example 2 Assessing the Detection Limit

Spiked samples were prepared using 0.001, 0.01, 0.1 or 1 ng of anthraxDNA with 50 ng sludge DNA. These amplification assays were set up in anattempt to determine the detection limit of anthrax DNA in the presenceof an overwhelming amount of non-specific background DNA. Typically, inRAPD assays, low stringency annealing conditions are used to allowpriming event even if the primers are not fully complementary to thepriming sites, i.e., a certain level of mis-match is allowed. In aneffort to determine which primers are more specific to the primingsites, an annealing temperature of 52° C. was used to preclude primingevents from mismatched sites. As shown in FIG. 5, primer nos. 173 and280 were able to amplify at high-stringency temperature even in thepresence of 0.001 ng (1 pg) control anthrax DNA. This result indicatesthat the PCR assay is highly sensitive for detection of anthrax DNA evenin the presence of 5,000-fold excess of non-specific DNA. Assuming 0.28pg dry wt./bacterial cell and 3% of the dry wt to be DNA (cited inNeidhardt, FC, ed., In: Escherichia coli and Salmonella, cellular andmolecular biology, ASM Press (1996)), the ability of a PCR-basedtechnology to detect the presence of 1 pg DNA suggests that thistechnology would detect DNA from as many, or as few, as 120-150 B.anthracis cells. While it is a most desirable objective of the inventionto be able to detect the presence of even fewer (preferably 1-10)anthrax cells, no other technology to date has been demonstrated to beas sensitive for anthrax detection as the PCR-based procedures justdescribed.

Example 3 Design and Use of High-Fidelity Primers

High-fidelity forward and reverse primer sets (15-20-bases in length)were designed based on the sequence of the cloned regions. Six sets ofprimers, F1-B1 (290R), F1-B20 (290F), F2-B12 (280R), F1-B22 (173R),F1-B17 (290F), and F1-B2 (248F) were selected to amplify fragmentsranging in length from 144-520 bp. Total DNA isolated from environmentalsources, soil and sludge, was spiked with anthrax DNA. Control DNAsamples contained 1.65-1.85 ng non-specific DNA and spiked samplescontained 1 pg of anthrax DNA. The ratio of non-specific DNA:anthrax DNAwas about 1,600:1. As shown in FIG. 6, except for the primer setcombination F1-B2, the other five primer sets amplified expectedfragments in spiked soil/sludge samples. This result demonstrates thateven in the presence of overwhelming non-specific DNA, the designedhigh-fidelity primers of the invention amplify anthrax-specificfragments.

The principles, preferred embodiments and modes of carrying out thepresent invention have been described in the foregoing specification.The invention which is intended to be protected herein, however, is notto be construed as limited to the particular forms disclosed, sincethese are to be regarded as illustrative rather than restrictive.Variations and changes may be made by those skilled in the art withoutdeparting from the spirit of the invention. For example, the pairs ofprimers described are merely representative of a small number of primersthat are specifically designed from the five fragments of anthraxchromosomal DNA. Numerous other primer sets and probes derivable fromthe inventive anthrax chromosomal DNA fragments are within the scope ofthe protection sought.

15 1 985 DNA Bacillus anthracis 1 gcctacgact cactataggg cgaattgggccctctagatg catgctcgag cggccgccag 60 tgtgatggat atctgcagaa ttcggcttatcaagctgctc tactaaatat tggagagctt 120 cgtcagacaa ccttacacct tctgtatatgcttctaattc tgccttcgat tcctcttctg 180 ttaattcact aatttctatt ttgttttccccaaacatatt tggtttatat ggaaaatgat 240 tttgcatcgt caccgcatga ataaatgtaggttgttttct cttttttaat tcatctatta 300 tttctttact catagataaa tcaccgatattatctccaac tacttctttg tttttcatcg 360 tatcttctgc attaaaatta tcgaatcctaatgttttata tacatcttcc cgtttataaa 420 acgatcggtt aaaagcatgg attgcacttgcataatatcc ttgtttcttt aattcacttg 480 caactgatgg gatttctttc tgacttggaatagcttgctg atacggtata gaacctggca 540 ttaaaagact cattgagtaa ctcgttaatgcttcaaattc tgtgtttgct gtattccctc 600 caaatgtagg agctatcgtt tgtccccctgggaaattctc tgtataacga tgtaaattcg 660 gcactggatc ttcgctaaat gaaagattcgttagttttgt cggatcccaa gaagcttcac 720 tcataataaa tataatattt ggcttctctacttttgtttg tgcctttact ttaccgctat 780 attgtttttc tatatccttt gcaatttgctgcatattttc ttttgaatat ccctctggct 840 ctttaataac tgtcgtatct aagttacttaaaaatccaat aataaaacca ttcgctttat 900 aatttgcagc ttgataagcc gaattccagcacactggcgg ccgttactag ggatccgagc 960 tcggtaccta gcaagggtat caaag 985 2710 DNA Bacillus anthracis 2 ggcgaattgg gccctctaga tgcatgctcg agcggccgccagtgtgatgg atatctgcag 60 aattcggctt caggcggcgt tggagcaggc tttgtgaacattattatgta tgcgattatc 120 gcagtcttta tatctggatt aatggtcgga cggacaccagagtttttagg taagaaaatt 180 gaaggtaagg aaatgaaatt aattgcggta acgatactatttcatccatt gctcatttta 240 ggattttcgg cattagctct ttcaacaagt ttagggacggatgctatttc tcattccggt 300 ttccacggtt taacgcaagt tgtatatgaa tatacatcgtcagctgcgaa taacggatct 360 ggatttgaag gattaggaga taatacaccg ttttggaatattacgactgg tttagttatg 420 tttttaggac gctacttcag tttaattacg atgctagctgtggcagcttc gctaaaagag 480 aaaacggttg taccagaaac agttggaacg ttccggacagataatggttt atttggaggc 540 atctttattg ggacgattgt aattgttggt gccgttagcattcttcccaa tgttagtact 600 cggaccaatt gcagaatttc ttacattgaa gtaatggagggtaaatgatg agaccggtag 660 tagtaaaaga aaaaagagtt aatgagtcac acatacatgccggtagaaga 710 3 722 DNA Bacillus anthracis 3 gactcctata gggcgaattgggccctctag atgcatgctc gagcggccgc cagtgtgatg 60 gatatctgca gaattcggccgcgagcactc tataaagcac aacaatgggt tgatactcac 120 agtccagaag agattgctgatgccgtttct ccgttattta aagacacttc aaaagacatt 180 acagaaaaag taattgaacggtataaaaag caacattctt atgcgacaaa tccgctatta 240 gatgctgaag aatggaaacagctccaaacg attatgaaag aagctggcga attacaaaaa 300 gaagttccac atgaagcgctcgtcaataca aaaattgccg agagcgttat taagaaatag 360 aggcgaagtg tatgagctttttacaaatac gtaacgtttc tcactgcttt ttcgcaaaag 420 agaatgccaa gctgattctcgaaaatatga gtttacaagt ggaagaaggc gaattcattt 480 ctatacttgg tccaagtggttgcggcaaaa cgacactcct ctccattatt gccgggctgc 540 ttgatccaat tgaaggtatcgtctttttag atggtgagcc cattacaacg aaaacttcat 600 ctatggggta tatgttgcagcaaggactac ttgtttcctt ggaagacaat tgaagaaaat 660 attatgctcg gacttcatatccgaaaaatt tacgatgaac agacgaaaga acatacttta 720 ca 722 4 754 DNABacillus anthracis 4 ctaggtaccg agctcggatc cctagtaacg gccgccagtgtgctggaatt cggcttcagg 60 cggcgtcaca acaacgatta agaaaataag agttaagctcgttaatactg tatttaaagc 120 aatctcattc ggtgtttttt gacgagcagc tccttctactaacgaaatca ttttatcaat 180 aaaggattta ccaggattac tcgtaatgac aatcgtaatctcatcactta caaccatcgt 240 accgcctgtt acggaacaaa aatcaccgcc cgcttcttttattacaggtg ctgattcccc 300 tgtaatcgca gattcatcaa cagacgctaa tcctttaatgacttcaccat cacttggaat 360 catctcacct tgctttacaa taacaacgtc atcttttttaaggtcagttg ctgaaacttg 420 aacaatttct ccattttctt ttacaacatt tgcaaacacatctttcttcg actgctttaa 480 agaatcagct tgcgctttac cacgaccttc tgctaatgcttctgcaaagt tcgcaaataa 540 aactgtaaat aatagaatga gagaaacagt tatattaaaccatcctggta tactactaga 600 atgacttgga agaaacgata aaatgaacgt aatgacaaacccaatttcta caacgaacat 660 aatcggattc tttatcataa ctttcggatt caatttcgcaacggattgtt tcatcgcatg 720 tttcacgata tcacgatcca tcgttttcgc ttgg 754 5932 DNA Bacillus anthracis 5 ctacgactcc tatagggcga attgggccct ctagatgcatgctcgagcgg ccgccagtgt 60 gatggatatc tgcagaattc ggcttgagta agcggtatgataaatgtata acaactagaa 120 aggagtggta aaacaaaatg tcactagaag cgctcattattttttctttg ctaaatgcgg 180 gtcagcttgc agagaattcg aaggtggatg tacataaagagcagaaagat gcttatgtat 240 atgttcagaa agaggaaaat aaataacttt attttatatataaacgaaaa aagccaatcc 300 acaggattgg ctttttgtca ttaacgagag tagaactctacgattaatgc ttcgttgatt 360 tcagctggta actcagcgcg ctcagcatga cgagtgtaagtagcttctaa tttgtcagca 420 tcgaaagtta agtattctgg tacgaagttg ttaacttcgatcgcttcttt aacaacaaca 480 aggttgttag atttttcgcg aacgctgata gtttgaccaggttttacacg gtaagatggg 540 atatctacgc gagcaccatc aaccatgatg tgaccgtggtttactaattg gcgagctgca 600 cgacgagtgc gagctaagcc catacggtaa actaagttgtcaagacgagc ttcaagaagg 660 atcatgaagt tttcgccgtg cttaccaggc attttacctgcttggtcaaa tgtgcgacgg 720 aattgacgct cagtcatgcc gtacatgtga cgaagtttttgtttctcttg taattgtaaa 780 ccgtattctg aaagtttctt acgttggtta ggaccgtgaggacctggtgc gtaagggcgt 840 ttttctaatt cttttcctgt gccgcttact caagccgaattccagcacac tggcggccgt 900 tactagtgga tccgagctcg gtaccaagca gg 932 6 440DNA Bacillus anthracis 6 gcctacgact cactataggg cgaattgggc cctctagatgcatgctcgag cggccgccag 60 tgtgatggat atctgcagaa ttcggcttat caagctgctctactaaatat tggagagctt 120 cgtcagacaa ccttacacct tctgtatatg cttctaattctgccttcgat tcctcttctg 180 ttaattcact aatttctatt ttgttttccc caaacatatttggtttatat ggaaaatgat 240 tttgcatcgt caccgcatga ataaatgtag gttgttttctcttttttaat tcatctatta 300 tttctttact catagataaa tcaccgatat tatctccaactacttctttg tttttcatcg 360 tatcttctgc attaaaatta tcgaatccta atgttttatatacatcttcc cgtttataaa 420 acgatcggtt aaaagcatgg 440 7 268 DNA Bacillusanthracis 7 cactcataat aaatataata tttggcttct ctacttttgt ttgtgcctttactttaccgc 60 tatattgttt ttctatatcc tttgcaattt gctgcatatt ttcttttgaatatccctctg 120 gctctttaat aactgtcgta tctaagttac ttaaaaatcc aataataaaaccattcgctt 180 tataatttgc agcttgataa gccgaattcc agcacactgg cggccgttactagggatccg 240 agctcggtac ctagcaaggg tatcaaag 268 8 22 DNA Bacillusanthracis 8 cattcggtgt tttttgacga gc 22 9 24 DNA Bacillus anthracis 9ctttgcagaa gcattagcag aagg 24 10 24 DNA Bacillus anthracis 10 tgttccaagaatgaagcgta ctcc 24 11 22 DNA Bacillus anthracis 11 tgaagcctac tcccgtttcaag 22 12 22 DNA Bacillus anthracis 12 tcaccgttag aatcacgcca cc 22 13 22DNA Bacillus anthracis 13 gccaaaacat ttatcgtccc ag 22 14 25 DNA Bacillusanthracis 14 caatgggttg atactcacag tccag 25 15 25 DNA bacillus anthracis15 ccttgctgca acatataccc catag 25

What we claim is:
 1. An isolated and purified DNA fragment fromchromosomal DNA of B. anthracis consisting essentially of the nucleotidesequence of SEQ ID NO:
 6. 2. An isolated pair of forward and reverseoligonucleotide primers for use in the amplification-based detection ofB. anthracis, each of said forward and reverse primers consisting of atleast 20 to 30 contiguous nucleotides of SEQ ID NO: 6 and optionallyincluding a detectable label, and wherein said forward and reverseprimers specifically amplify B. anthracis DNA and do not amplify DNAfrom related strains of B. cereus, B. thuringienis, and B. subtilis. 3.The forward and reverse primers according to claim 2, wherein saidprimers do not include said optional detectable label.
 4. An isolatedoligonucleotide probe for use in hybridization-based detection of B.anthracis, said probe consisting of a fragment of at least 30nucleotides from the nucleotide sequence of SEQ ID NO: 6 whichspecifically binds complementary strand DNA from B. anthracis and doesnot bind DNA from related strains B. cereus, B. thuringiensis, and B.subtilis, and said probe optionally including a detectable label.
 5. Theoligonucleotide probe according to claim 4, wherein said probe is boundto a solid support.
 6. The oligonucleotide probe according to claim 4,wherein said probe does not include said optional detectable label.
 7. Amethod for detection of B. anthracis in an environmental samplecontaining non-specific DNA, comprising the steps of: (a) providing apair of primers, wherein each of said primers consist of at least 20 to30 contiguous nucleotides of SEQ ID NO: 6 and wherein said pair ofprimers specifically amplify B. anthracis DNA and do not amplify DNAfrom related strains of B. ceures, B. thuringienis, and B. subtilis, andwherein said primers optionally include a detectable label; (b) mixingsaid primers with DNA isolated from said environmental sample; (c)amplifying any DNA to which the primers in step (b) anneal by use ofpolymerase chain reaction; and (d) detecting any B. anthracis DNA insaid environmental sample based an the amplification products of step(c).
 8. The method of claim 7, wherein said pair of prime do not includesaid optional detectable label.
 9. A method for detection of B.anthracis in an environmental sample, comprising the steps of: (a)providing at least one oligonucleotide probe, wherein said probeconsists of a fragment from the nucleotide sequence of SEQ ID NO: 6 andwhich specifically binds complementary strand DNA from B. anrthracis anddoes not bind DNA from related Strains B. cereus, B. thuringiensis, andB. subtilis, said probe optionally including a detectable label; (b)conjugating said probe to a solid support; (c) contacting saidenvironmental sample with said support-bound probe formed in step (b)under conditions favorable for hybridization; and (d) detecting any B.anthracis DNA in said sample based on the hybridization products of step(c).
 10. The method of claim 9 wherein said probe does not include saidoptional detectable label.
 11. A kit for the detection of B. anthracis,comprising: (a) a carrier to receive therein one or more containers; and(b) at least one of said containers including a pair of oligonucleotideprimers wherein each of said primers consist of at least 20 to 30contiguous nucleotides of SEQ ID NO: 6 and wherein said pair of primersspecifically amplify B. anthracis DNA and do not amplify DNA fromrelated strains of B. cereus, B. thuringienis, and B. subtilis, saidpair of primers optionally including a detectable label.
 12. The kit ofclaim 11, wherein said pair of primers does not include said optionaldetectable label.
 13. The kit of claim 11, further comprising a secondcontainer containing primers for amplifying the TOX gene and the CAPgene of B. athracis.
 14. A kit for the detection of B. anthracis,comprising: (a) a carrier to receive therein one or more containers; and(b) at least one of said containers including an isolatedoligonucleotide probe, wherein said probe consists of a fragment of atleast 30 nucleotides from the nucleotide sequence of SEQ ID NO: 6 andwhich specifically binds complementary strand DNA from B. anthracis anddoes not bind DNA from related strains B. cereus, B. thuringensis, andB. subtilis, said probe optionally including a detectable label.
 15. Thekit of claim 4, wherein said probe does not include said optionaldetectable label.
 16. The kit of claim 14, further comprising a secondcontainer containing primers for amplifying the TOX and the CAP gene ofB. anthracis.